Bromelain is a mixture of proteinases derived from pineapple stem that is sold in health food stores as a "digestive aid". Beneficial therapeutic effects of bromelain have also been suggested or proven in several human inflammatory diseases and animal models of inflammation. Self-treatment with oral bromelain induced clinical and endoscopic remission of ulcerative colitis in two patients whose disease was refractory to conventional multi-agent medical therapy. Our controlled animal studies show that bromelain treatment decreases both the development of spontaneous colitis and the severity of established colitis in the IL-10 knockout (IL-10 -/-) murine model of inflammatory bowel disease (IBD). The biological effects of bromelain appear to depend on its proteolytic activity, which can vary from lot to lot according to its proteinase composition. It is critical that the bromelain to be used in human trials contain appropriate levels of active ingredients. However, it is not currently known which proteinases within bromelain account for its anti-inflammatory activity in IBD. We have developed assays to specifically measure the proteolytic activity of the 3 major bromelain proteinases (stem bromelain, ananain, and fruit bromelain) as well as procedures to purify these enzymes. In this study, IL-10-/- mice with established colitis will be treated orally with purified bromelain proteinases and effects on colonic inflammation will be determined. The proposed studies will definitively determine how the 3 major bromelain proteinases contribute to anti-inflammatory effects of bromelain within the colon. They will also begin to determine the mechanisms by which this occurs. This information will facilitate further investigations of the mechanisms involved as well as the rational design of future clinical trials of this promising complementary colitis therapy. They will thus strengthen the scientific knowledge base needed to inform the practice of complementary and alternative medicine.